Sacccharomyces cerevisiae has proven versatile as a host species for the expression of foreign polypeptides. Many different proteins from a variety of species have been expressed in S. cerevisiae, some to levels of &gt;10% of total cell protein. Typically, expression has been mediated by a plasmid containing yeast regulatory sequences (transcriptional promoter and terminator) circumscribing the structural gene for the expressed polypeptide as well as other sequences required for the selection and amplification of plasmids in both S. cerevisiae and in Escherichia coli. In addition, it has been possible to integrate the yeast regulatory sequences circumscribing the structural gene for the expressed polypeptide into a yeast chromosome and achieve high-level expression.
S. cerevisiae has 4 genes which encode the enzymes responsible for the utilization of galactose as a carbon source. The GAL1, GAL2, GAL7, and GAL10 genes respectively encode galactokinase, galactose permease, .alpha.-D-galactose-1-phosphate uridyltransferase, and uridine diphosphogalactose-4-epimerase. In the absence of galactose, very little expression of these enzymes is detected. If cells initially are grown on medium containing glucose, and galactose is added to the culture, these four enzymes are induced coordinately by at least 1000-fold upon depletion of glucose from the media. This induction has been shown to occur at the level of RNA transcription. The GAL1, GAL2, GAL7 and GAL10 genes have been molecularly cloned and sequenced. The regulatory and promoter sequences to the 5' sides of some of the respective coding regions have been placed adjacent to the coding regions of the lacZ gene. These experiments have defined those promoter and regulatory sequences which are necessary and sufficient for galactose induction and which are useful for driving the expression of heterologous genes.
The GAL4 and GAL80 gene products are respectively positive and negative regulators of the expression of the GAL1, GAL2, GAL7, and GAL10 genes as well as of the MEL1 gene, which encodes .alpha.-galactosidase, an enzyme required for melibiose catabolism. The GAL4 product acts as a positive regulator at the transcriptional level by binding to specific DNA sequences 5' to the structural information of these genes. In the absence of galactose, the GAL80 gene product (protein) interacts with the GAL4 gene product or with a DNA sequence to prevent this transcriptional activation event. In the presence of galactose, the GAL80 protein apparently cannot antagonize the GAL4 protein, and the GAL4 protein can function as a transcriptional activator. The GAL4 gene is expressed at very low levels, and its product is rate-limiting for the optimal induction of the GAL gene promoters. This is especially true when a cell contains multiple copies of plasmids with galactose-inducible promoters driving the expression of heterologous genes. However, if it is a goal to maintain minimal or no expression of a heterologous gene before the addition of galactose, it is important that the GAL4 gene be expressed at very low levels in the absence of galactose.
In a variety of recombinant microbial expression systems, the synthesis of many different heterologous polypeptides has been shown to be deleterious to the host cell. As a consequence, there is selective pressure against the expression of such heterologous polypeptides, such that the only cells which accumulate in a scale-up of such a recombinant culture are those which do not express the heterologous polypeptide or express so little of the heterologous polypeptide that the culture becomes an uneconomical source of that polypeptide. An optimal scheme for the scale-up of such a recombinant culture would be to maintain minimal or no expression of the heterologous gene during the expansion of the culture to a large volume and high cell density and then to induce the maximal expression of the heterologous gene only in the final stage of culture growth prior to product isolation.